Active substance of morchella, its use and a composition thereof for improving the reproductive function

ABSTRACT

The present invention provides an active substance of  Morchella , its use and a composition thereof for improving disturbance of reproductive function, especially, for manufacturing a pharmaceutical composition to improve disturbance of reproductive function induced by obesity or metabolic syndrome. The composition with the active substance of  Morchella  can effectively improve the structural integrity of testicular tissues and sperm, increase testosterone levels in the blood, and reduce the oxidation stress in sperms.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to an active substance of Morchella forimproving reproductive function, its use and composition thereof; morespecifically, to a composition of Morchella for increasing testosteronelevels, reducing the proportion of abnormal sperm, reducing oxidativestress in sperms, and improving structural integrity of testiculartissue induced by obesity or metabolic syndrome.

Description of the Prior Art

A reproductive function can be evaluated from different aspects, suchas, but not limited to, the structural integrity of the testiculartissue, the number of abnormal sperms, the concentration of testosteronein the blood, or the oxidative stress in sperms.

Testis

A testis consists of several seminiferous tubules, which are supportedby sertoli cells and separated by groups of Leydig cells.Spermatogenesis is regulated by sertoli cells and Leydig cells, and theconcentration of androgens is also maintained by these cells to improvesperm maturation (Kotaja et al., 2004). From the aspect of tissuestructure, immature sperm cells are located in the basal membrane of theseminiferous tubules and move to the luminal center of the seminiferoustubules as the sperms mature.

Testosterone

Testosterone is a steroid hormone derived from cholesterol, which issecreted mainly by a testis of a male or an ovary of a female.Statistics show that testosterone secretion of an adult male is about 20times of that of an adult female, and the content of testosterone in anadult male is 7-8 times of that of an adult female.

Testosterone is the main male hormone in human bodies. Althoughtestosterone tends to produce noticeable symptoms in males than females,it is equally important to women. Therefore, for both of men and women,testosterone has a significant impact on physical activities in humanbodies. The benefits of testosterone include increasing libido,strength, stimulating the immune system, and preventing osteoporosis.Since testosterone can improve physical strength, some athletes haveingested testosterone before games to stimulate and optimize theirconditions. However, at present, testosterone is banned in most sports.

After the age of 40, the concentration of circulating testosterone fallsevery year and would lead to a diagnosis of testosterone deficiencysyndrome. This symptom is common in middle-aged and elderly men and isgenerally considered to be related to menopause. Although male menopausedoes not occur at a pronounced time as female menopause, symptoms ofmale menopause gradually become severe as the concentration oftestosterone decreases. Common symptoms of male menopause include lossof libido, erectile dysfunction, diminished vitality, restlessness,depression, loss of enjoyment of life, decreased concentration,decreased work efficiency, and sleep disorders. In addition, thesymptoms are accompanied by degeneration of secondary sexualcharacteristics, osteoporosis, increased fat, testicles shrinking, andgynecomastia. In addition to the above-mentioned symptoms, which affectthe quality of life, many studies have found that the risks ofosteoporosis, fractures, and cardiovascular diseases increase when thereis not enough testosterone in a human body. Moreover, when serumtestosterone levels in men are down, so is the concentration of spermand/or the total number of sperms in semen, decreasing the probabilityof conception.

Another factor that influences testosterone levels is obesity. Previousstudies have found that the total testosterone concentrations are lowerin obese patients than in men with normal BMI, suggesting an inversecorrelation between testosterone levels and BMI. Besides, almost one inevery four obese people has a significantly lower concentration oftestosterone in the blood. The mechanism underlying for this effect isthat the aromatase enzyme in adipocytes catalyzes the conversion oftestosterone to estrogen. Therefore, it is believed that a higherconcentration of estrogen and a lower concentration of testosterone arein the blood of obese men due to the increase in available aromataseenzyme in the fatty tissue. In addition, the above-mentioned enhancedcatalysis becomes more pronounced with age. However, research has alsoshown that obese individuals can restore normal testosterone levelsthrough weight loss and exercise.

The concentration of testosterone in the blood of patients withmetabolic syndrome is also relatively low compared to healthy males.Corona G et al. published a study on diabetes in people with metabolicsyndrome (Type 2 diabetes mellitus and testosterone: a meta-analysisstudy) in 2010. This study demonstrated that there is a lowerconcentration of testosterone in the blood of type 2 diabetic patients,and points out that erectile dysfunction in patients with type 2diabetes become worse when the concentration of testosterone decreases.Furthermore, Adamopoulos et al. (1987) and Jelodar et al. (2009)reported that diabetes mellitus can cause insulin resistance andinflammation, thereby inhibiting the release of hormone from thepituitary gland and production of testosterone from the Leydig cells inseminiferous tubules.

Oxidative Stress

Growing evidence indicates that oxidative stress is increased indiabetes and has deleterious effects on male reproductive function(Amaral et al., 2008). Oxidative stress can induce DNA fragmentationduring sperm transportation or storage through the epididymis. Comparedwith other tissues, sperms contain more unsaturated fatty acids andtherefore are more susceptible to ROS attack, resulting in sperm DNAfragmentation (Parlaktas et al., 2008).

Morchella

Morchella is a world-famous large edible fungus, belonging toAscomycetes, Pezizales, Morchellaceae, Morchella. Morchella is named forits irregular folds, which looks like goat tripe in the upper part ofits fruiting bodies. Morchella is also known as goat tripe mushroom,goat tripe vegetables, clogs bacteria, morel, etc. Bunyard et al.published a study in 1994 and divided Morchella into at least two groupsby RFLP analysis of the 28S RNA gene: (1) black Morchella, whichincludes M. angusticeps, M. conica, and M. elata, with darker browncolor at the head and a regular grid-like pattern of folds; (2) yellowMorchella, which includes M. deliciosa, M. esculenta, and M. crassipes,with a pale yellow to brown color at the head and irregular folds.

Morchella is widely distributed over all continents, especially in thetemperate regions of the northern hemisphere, except the north and southpoles. Its growing season is in late spring or early summer. AsMorchella can only be harvested once a year, the prices of Morchellaremain high due to limited production. However, Morchella is popularbecause of its tasty flavor and its rich nutritional value, as shown bythe earliest record of artificial cultivation of Morchella in the 1880s.Nevertheless, the technology of artificial cultivation of fruitingbodies of Morchella has not yet been fully mastered. Although there hasbeen semi-commercialized production in China, it is still limited due tomany conditions. Therefore, the fermentation technology of mycelia ofMorchella emerged. The advantages of Morchella growing using liquidfermentation is that the fermentation parameters can be controlled,which lead to rapid growth of the mycelia, and thereby reduce the riskof contamination as the growth period is short. In addition, mycelia ofMorchella are tastier than its fruiting bodies, which create fewerbarriers in product development.

Morchella is also extremely attractive in terms of health care.Morchella has been used as an edible mushroom since ancient times inChina, and its records can be traced back to “Compendium of MateriaMedica” of the Ming Dynasty. The article states that goat tripevegetables (i.e. Morchella) is “cold and non-toxic, beneficial to thestomach, phlegm dispelling, and Qi regulating,” which shows thatMorchella has a certain physiological activity. In recent years,research has also proved the efficacy of Morchella by scientificmethods, including anti-tumor, kidney protection, anti-fatigue, liverprotection, anti-radiation, antibacterial, regulating immunity, andregulating gastrointestinal motility.

The present invention is the first study to demonstrate the effect ofMorchella mycelia on sexual dysfunction induced by hyperglycemia,specifically, by administering the active substance of Morchella toincrease the concentration of testosterone in blood, reduce theproportion of abnormal sperms, decrease oxidative stress of sperms,and/or improve type of testis tissue. The present invention providesanother exploitable use of Morchella in the medical field, significantlyenhancing the value of Morchella as a pharmaceutical composition.

SUMMARY OF THE INVENTION

The object of the present invention is to develop an active substance ofMorchella for improving disturbance of reproductive function, preparedby the following methods:

-   -   (A) inoculating Morchella mycelia on a medium for solid-state        culture;    -   (B) inoculating the Morchella mycelia cultured in step (a) in a        liquid medium for liquid culture; and    -   (C) inoculating the Morchella mycelia cultured in step (b) in a        fermenter for fermentation.

Preferably, the temperature of step (c) is 23 to 28° C., the ventilationis 0.5 to 1.0 vvm, the rotation speed is 30 to 50 rpm, and/or the numberof cultivation day is 6 to 10 days.

Preferably, the Morchella is deposited in the Food Industry Research andDevelopment Institute, with a deposit number BCRC-36352 and/orBCRC-36336.

Another object of the present invention is to develop a method forimproving disturbance of reproductive function, comprising an effectiveamount of an active substance of Morchella.

Preferably, the Morchella comprises Morchella esculenta, Morchellacrassipes, or a combination thereof.

Preferably, the active substance of Morchella is a lyophilized powderobtained after fermentation and freeze-drying of Morchella mycelia.

Preferably, the active substance of Morchella is an extract obtained byextracting Morchella mycelia with pure water or ethanol.

Preferably, the reproductive function comprises testosterone level,sperm type, oxidative stress in sperm, structural integrity oftesticular tissues, or a combination thereof.

Preferably, improving the disturbance of reproductive function isincreasing the testosterone levels in the blood.

Preferably, improving the disturbance of reproductive function isreducing the proportion of abnormal sperms with broken tails, benttails, or bent necks.

Preferably, improving the disturbance of reproductive function isreducing the amount of ROS produced in sperms to reduce the oxidativestress in sperms.

Preferably, improving the disturbance of reproductive function isreducing the number and volume of gaps within the luminal center ofseminiferous tubules or between the seminiferous tubules to improve thestructural integrity of a testicular tissue.

Preferably, the disturbance of reproductive function is induced byobesity and/or metabolic syndrome.

Preferably, the metabolic syndrome is diabetes.

The present invention provides the benefits of Morchella in the medicalfield and further provides a composition for improving disturbance ofreproductive function, which comprises an effective amount of an activesubstance of Morchella.

Preferably, the active substance of Morchella is a lyophilized powderobtained after fermentation and freeze-drying of Morchella mycelia.

Preferably, the active substance of Morchella is an extract obtained byextracting Morchella mycelia with pure water or ethanol.

Preferably, the effective amount is 500 mg/60 kg to 10 g/60 kg bodyweight/day.

Preferably, the composition further comprises an additive selected fromthe group consisting of an excipient, a preservative, a diluent, afiller, an absorption enhancer, a sweetener, and a combination thereof.

Preferably, the composition is a medicine, feed, drink, nutritionalsupplement, dairy product, food, or health food.

Preferably, the composition is in the form of powder, lozenge,granulation, microcapsule, ampoule, liquid spray, or suppository.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the stained sections of testis tissues of the control group(C), the diabetic group (D), the diabetic group administered Morchellaesculenta (D+ME), and the diabetic group administered Morchellacrassipes (D+MC).

FIG. 2 shows the type of sperms of the control group (C), the diabeticgroup (D), the diabetic group administered Morchella esculenta (D+ME),and the diabetic group administered Morchella crassipes (D+MC).

FIG. 3 shows the content of testosterone in plasma of the control group(C), the diabetic group (D), the diabetic group administered Morchellaesculenta (D+ME), and the diabetic group administered Morchellacrassipes (D+MC).

FIG. 4 shows the ROS content in sperms of the control group (C), thediabetic group (D), the diabetic group administered Morchella esculenta(D+ME), and the diabetic group administered Morchella crassipes (D+MC).

DETAILED DESCRIPTION OF THE EMBODIMENTS

Source of Fungus

Morchella can be selected from the group consisting of M. angusticeps,M. conica, M. elata, M. deliciosa, M. esculenta, and M. crassipes. In apreferred embodiment, two Morchella species are purchased from theBioresource Collection and Research Center (BCRC) of the Food IndustryResearch and Development Institute. One is Morchella esculenta, with adeposit number BCRC-36352, the other is Morchella crassipes, with adeposit number BCRC-36336. The active substances of Morchella of thepresent invention are not limited to the ones obtained by theabove-mentioned species.

Fungus Cultivation

Mycelia of the purchased Morchella were inoculated on the solid mediumto activate the fungus. In a preferred embodiment, the solid medium isPotato dextrose agar (PDA). After the growth of mycelia of Morchella wascomplete, a cube of fresh mycelia together with the solid culture mediumwas inoculated into a flask containing 1 L of the liquid medium. In apreferred embodiment, the formulation of the liquid medium is shown inTable 1. In a preferred embodiment, the mycelia are liquid culturedunder the conditions of a temperature of 25° C. and a speed of 50 to 150rpm for 4 to 7 days to complete the growth of the mycelia.

TABLE 1 Formulation of the liquid medium is shown as follows:Composition Proportion Sucrose    1-10% Yeast extract, YE   0.1-2%Soybean powder    1-10% KH₂PO₄ 0.01-0.2% MgSO₄ 0.01-0.2%

Subsequently, the mycelia growing completely in the flask wereinoculated in a sterilized medium in a 100-liter fermenter for massproduction. In a preferred embodiment, the mycelia of Morchella aremass-cultured at a temperature of 25° C., a ventilation of 0.5-1.0 vvm,and a rotational speed of 40 rpm for 6 to 10 days. When the dry weightof the mycelia stops increasing or when the sucrose residue was lessthan 1000 mg/L, the fermenter was heated to stop cultivation. In apreferred embodiment, the fermenter was heated to 100° C. for 15 minutesto stop cultivation.

Preparation of Lyophilized Powder

After fermentation was completed, a part of the whole fermented liquid(containing the culture medium and the mycelia) in the fermenter wasspread on a plate and freeze-dried to prepare a “lyophilized powder ofwhole fermented liquid.” In a preferred embodiment, the freeze-dryingtemperature is set at 30° C. and the freeze-drying duration is at least3 days. The other part of the whole fermented liquid was centrifuged,and the mycelia of Morchella were freeze-dried to obtain “lyophilizedpowder of mycelia.” In a preferred embodiment, the whole fermentedliquid was centrifuged at 4500 rpm by using a continuously centrifugaldecanter centrifuge.

Preparation of Extract

An appropriate amount of “lyophilized powder of whole fermented liquid”was resuspended in pure water. In a preferred embodiment, the volume ofpure water for resuspending is 5 to 40 times, more preferably 20 times,of the weight of the lyophilized powder. Subsequently, the mixture washeated at 121° C. for 20 minutes, and then the mycelia were removed bycentrifugation (4500 rpm, using a continuously centrifugal decantercentrifuge) to obtain an extract of whole fermented liquid. The extractof whole fermented liquid was lyophilized to obtain “hot water extract.”An appropriate amount of “lyophilized powder of mycelia” was resuspendedwith alcohol. In a preferred embodiment, the volume of the alcohol forresuspending is 5-40 times, more preferably 20 times, of the weight ofthe lyophilized powder. In a preferred embodiment, the concentration ofalcohol is 95-100%. In a preferred embodiment, the alcohol is ethanol.

The resuspended lyophilized powder of mycelia was sonicated for 2 hoursand then centrifuged (4500 rpm, using a continuously centrifugaldecanter centrifuge) to obtain mycelium extraction. The myceliumextraction was concentrated under reduced pressure to obtain “ethanolextract.” The hot water extract and the ethanol extract were thoroughlymixed to obtain an “extract mixture” as an active substance forMorchella feeding tests. In a preferred embodiment, the hot waterextract and the ethanol extract are mixed in a ratio of 1:1 by weight.

Test Animals

Male 5-week old Sprague Dawley (SD) rats were purchased from BioLASCOTaiwan Co., Ltd. The transparent cages for the experimental rats weresterilized with 75% ethanol before the rats were put in. Two rats werehoused in a cage at a temperature in the range of 23±1° C., withhumidity maintained at 40-60%, light and dark cycles of 12/12 (7:00 a.m.to 6:59 p.m. bright, and 7:00 p.m. to 6:59 a.m. dark). Feed pellets(Laboratory Rodent Diet 5001, PMI® LabDiet®, St. Louis, Mo., U.S.A.),feed, and distilled water were supplied ad libitum.

Animal Feed

Feed powder Laboratory Rodent Diet 5001M (PMI Nutrition International,Inc., USA) was purchased from Young Li Co., and lard is selected as fatand was purchased from High Science Co., Ltd., (Taiwan). The formulationis shown as follows:

Weight percentage (%) Control Group High-Fat Diet Group Crude protein23.9 21.5 Crude fat 5.0 18.4 Carbohydrates 53.8 40.8 Ash 7.0 6.0 Others10.3 13.3

The feed of the high-fat diet group was made by adding lard to the feedpowder, wherein the fat was 40% of total calories, to induce insulinresistance.

Test Groups

Male 5-week old Sprague-Dawley rats were housed for one week, with 8 ineach group, for a total of 32 animals in four groups. One group was thecontrol group while the other three groups were feed with the aboveanimal feed and injected with streptozocin (STZ) to create diabetesmodel rats. Out of three diabetic groups, two groups were administratedwith Morchella esculents and Morchella crassipes, respectively.

Name and abbreviation of each group are shown as follows:

-   -   1. Control group (C)    -   2. Diabetes group HFD/STZ-Diabetes (D)    -   3. Diabetes group feeding Morchella esculenta (D+ME)    -   4. Diabetes group feeding Morchella crassipes (D+MC)

Tube Feeding and Sacrifice

After diabetes was successfully induced in the experimental rats, therats were fed with samples by Tube Feeding. The extract mixture wasdissolved in a carrier solution and fed with an amount of 500 mg/kg bodyweight (B.W.) to the Morchella esculenta (D+ME) diabetic group andMorchella crassipes (D+MC) diabetic group. The control group wasadministered the same volume of vehicle solution (vehicle, 0.1 mol/Lcitric acid buffer, pH 4.5) as the volume administered to theexperimental groups. Body weights of the experimental animals weremeasured and recorded weekly. After four weeks of sample feeding, theexperimental animals underwent Oral Glucose Tolerance Test (OGTT). Afteranimals of each group were fasted for 12 hours, blood was collected fromtail vein, and concentration of blood glucose was measured. At the endof the experiment, carbon dioxide was used for anesthesia of the rats,syringe and centrifuge tube were pre-wetted with heparin (500 IU/mL),and blood was drawn from the abdominal aorta to sacrifice the rats. Theobtained blood was taken as whole blood, loaded into a 15 mL centrifugetube, and centrifuged at 3000×g for 15 minutes at 4° C. Aftercentrifugation, the supernatant was collected as rat plasma. At the sametime, the rats were sacrificed and the organs were removed and weighed.Liver, testis, and hypothalamus were stored at −80° C. for furtherexperimental analyses, while sperms in epididymis were analyzed on theday of sacrifice.

Sections and Staining of Testis

Rats were sacrificed, and their testes were immersed in 10% formaldehyde(formalin) for later use. The testis tissues fixed with formaldehydewere cut with a scalpel to obtain a thickness of 0.5 cm tissue.Afterward, the tissues were immersed in formaldehyde again. Stainedsections of the testis tissues were prepared by hematoxylin and eosin(H&E) staining. In hematoxylin and eosin (H&E) staining, hematoxylin isa basic dye for staining nucleus chromatin and cytoplasmic ribosomes apurplish blue, and eosin is an acid dye for staining the composition ofthe cytoplasm and extracellular matrix red. Therefore, based on thestaining results, the structure of the tissues can be determined. Afterthe sections of rat testis tissues were stained, the type of theseminiferous tubules of the testes was observed.

Collection of Sperms

Sperms in the epididymis were collected using a swim-up method. Theepididymis was removed from the sacrificed rats and placed in a beakercontaining 8 mL of RPM′ culture medium. Epididymis was cut in twosections with dissecting scissors, placed on a rocker to shake for 10minutes, and centrifuged at 190×g for 5 minutes. After centrifugation,the samples were placed in an incubator with 5% carbon dioxide at 37° C.for 30 minutes. Finally, sperms with better activity in the upper layerwere collected to observe the abnormalities of the sperms.

Determination of Testosterone Levels

Concentrations of testosterone in the serum of the plasma samples weretested by testosterone ELISA kits. 50 μL of rat plasma were added to 50μL of acetylcholinesterase (AChE) and anti-testosterone plasma forreaction. Next, the supernatant was removed and washed 5 times with awash buffer. Ellman's reagent was added as AChE substrate and shaken inthe dark for 80 minutes to complete the test sample. The absorbance ofthe sample at 412 nm was measured with an ELISA reader, and theconcentration of testosterone (ng/mL) in the serum was obtained byconverting the standard curve of the standard into the absorbance of thesample.

ROS Assay

DCFH-DA is a fluorescent probe that does not itself fluoresce. WhenDCFH-DA enters a cell, hydrolysis by esterase generates anon-fluorescent polar substance, DCFH, which is oxidized byintracellular ROS after it enters the cell, making it a fluorescent DCF,which can indirectly measure the amount of intracellular ROS production.

DCFH-DA was added to sperm RPMI solution containing 1×10⁶ sperms/mL byadjusting the obtained 1 mL sperm of each group to a final concentrationof 20 μM and incubated at 37° C. for 30 minutes. Then, the samples werecentrifuged at 760×g for 5 minutes. The supernatant was removed aftercentrifugation and washed once with PBS. The samples were centrifugedagain to remove supernatant, and 1 mL of PBS was added to resuspend thecells. The samples were analyzed by flow cytometry, which uses cellquest software. Ten thousand (10,000) cells were collected as a testsample, and the fluorescence intensity emitted by the machine was usedto estimate the amount of ROS in the cells of the test samples. A highabsorbance indicates a high level of ROS in the cells of the samples.

Statistical Analysis

Statistical analysis of the data was conducted using Statistical Product& Service Solutions (SPSS) software version 19.0, and the experimentalresults are expressed as Mean±S.E.M. Line charts were plotted using aPaired-Sample T-test with a statistical significance of p<0.01 (**) andp<0.05 (*). Histograms were plotted based on One-way analysis ofvariance One-way ANOVA and then using Duncan's Test for multiplecomparison methods, with p<0.05 representing statistically significantdifferences.

Based on the experimental results obtained from the above experiments,the details are described as follows.

The integrity of Testicular Tissues

Staining results of rat testis tissues are shown in FIG. 1. Asignificant number of enlarged lumen was observed within theseminiferous tubules of the diabetic group (D) as compared to thecontrol group (C) of the non-induced diabetic group, indicating that thenumber of sertoli cells in the seminiferous tubules of the diabeticgroup (D) was significantly smaller than that of the control group (C).In addition, there are greater gaps between the seminiferous tubules ofthe diabetic group (D), indicating that the number of Leydig cells ofthe diabetic group (D) was also smaller than that of the control group(C). Based on the experimental results from the control group (C) andthe diabetic group (D), suggesting that that diabetes induced by ahigh-fat diet can cause damage to the testicular tissues and candecrease a significant amount cells in the seminiferous tubules byenlarging lumen in and between the tissues.

In contrast, gaps within the luminal center of the seminiferous tubulesor between the seminiferous tubules of the diabetes groups feedingMorchella esculenta (D+ME) and Morchella crassipes (D+MC) aresignificantly fewer when compared to the diabetic group (D), indicatingthat these groups have more sertoli cells and Leydig cells. Based on theresults of the stained sections of the testicular tissues, the decreasednumber of sertoli cells and Leydig cells in the testis caused bydiabetes can be restored back to normal status by administration of theactive substance of Morchella, suggesting that Morchella has abeneficial effect on the male reproductive function.

Type of Sperms

The collected sperms are shown in FIG. 2. Diabetic group (D) has ahigher proportion of abnormal sperms than those in the control group(C). Examples of abnormal types include broken tails, bent tails, orbent necks of sperms. The two yellow arrows on the right on the photo ofdiabetic group (D) refer to sperms with bent necks, while the two yellowarrows on the left on the photo of diabetic group (D) refer to spermswith broken tails.

In contrast, sperm in diabetes groups feeding Morchella esculenta (D+ME)and Morchella crassipes (D+MC) have similar structures to those in thecontrol group (C), indicating that administrating the active substancesof Morchella can reduce structural damages on sperms caused by diabetes.

Testosterone Levels

The results of the testosterone levels in rat plasma samples are shownin FIG. 3. The testosterone levels in the plasma of the diabetic group(D) is less than about 0.25 ng/mL, whereas that of the control group (C)of the non-induced diabetic group is about 0.5 ng/mL, indicating thatthe testosterone levels in the plasma of the diabetic group (D) are lessthan that of the control group (C).

In contrast, the testosterone levels of two diabetes groups feedingMorchella esculenta (D+ME) and Morchella crassipes (D+MC) is about 1.0ng/mL and 1.25 ng/mL, respectively, which are both higher than that ofthe diabetic group (D). The testosterone levels in both D+ME and D+MCgroups were significantly higher when compared to that of the controlgroup (C). Based on these results, it is shown that administration ofactive substances of Morchella can stimulate increases in the lowsecretion or production of testosterone caused by diabetes.

ROS Content

The results of ROS content in sperms are shown in FIG. 4. Thefluorescence of the ROS generated by the control group (C) of thenon-induced diabetic group is defined as 100%. The fluorescence of theROS generated by the diabetic group (D) was significantly higher andabout 150% of that of the control group (C), which is 1.5 times of thefluorescence of the control group.

In contrast, ROS content of diabetes groups feeding Morchella esculenta(D+ME) and Morchella crassipes (D+MC) decreased to about 120% and 100%,respectively, which are similar to the content of the control group (C).Based on these results, it can be concluded that administration ofactive substances of Morchella can reduce the oxidative stress caused bydiabetes.

Based on the above experimental results, it was confirmed that theactive substance extracted from Morchella can increase the reproductivefunction of male animals, suggesting a novel use of Morchella in themedical field is developed. Therefore, a composition comprising theactive substance of Morchella can be manufactured and administered to anindividual in an effective amount to achieve a healing effect in thereproductive function.

As used herein, an “effective amount” refers to an amount that issufficient to produce the aforementioned prophylactic and/or therapeuticeffect. Based on in vitro cell culture experiments, the aforementionedeffective amount is defined as “μg/ml” based on the total volume of cellculture medium used in each culture. Based on animal model experiments,the aforementioned effective amount is defined as “g/60 kg bodyweight/day.” In addition, the data of effective amount obtained via invitro cell culture experiments can be converted to a reasonableeffective amount for animal use by the following formula:

1. In general (Reagan-Shaw et al., 2008), 1 “μg/ml” units (based on theeffective amount of in vitro cell culture experiments) may be equivalentto 1 “mg/kg body weight/day” units (based on the effective amount of ratmodel experiments), and, based on that metabolic rate of a rat is sixtimes of that of a human, the effective human dose can be found.

2. Therefore, an effective amount for use in mice based on an in vitrocell culture experiment of 500 μg/ml is calculated as 500 mg/kg bodyweight/day (i.e., 0.5 g/kg body weight/day). Further, taking intoaccount the differences in the aforementioned metabolic rates, aneffective amount for human use may be taken as 5 g/60 kg bodyweight/day.

3. Based on the test results reported above, a validated dose based on arat experiment is 500 mg/kg body weight/day and, therefore, a reasonablyeffective dose for human use should be 5 g/60 kg body weight/day.

In a preferred embodiment, an effective amount of the active substanceof Morchella contained in the composition is 500 mg/60 kg to 10 g/60 kgbody weight/day.

The composition further comprises an additive. In a preferredembodiment, the additive may be an excipient, a preservative, a diluent,a filler, an absorption enhancer, a sweetener, or a combination thereof.The excipient can be selected from sodium citrate, calcium carbonate,calcium phosphate, or a combination thereof. This preservative, such asbenzyl alcohol, parabens, extends the shelf life of pharmaceuticalcompositions. The diluent can be selected from water, ethanol, propyleneglycol, glycerol, or a combination thereof. The filler can be selectedfrom lactose, nougat, ethylene glycol of high molecular weight, or acombination thereof. Absorption enhancers may be selected fromdimethylsulfoxide (DMSO), laurocapram, propylene glycol, glycerol,polyethylene glycol, or a combination thereof. The sweetener may beselected from Acesulfame K, aspartame, saccharin, sucralose, neotame, ora combination thereof. In addition to the additives listed above, otheradditives that are suitable for use may be selected based onrequirements without affecting the medical effect of the activesubstance of Morchella.

The composition can be developed as a different product in the medicalfield. In a preferred embodiment, the composition is a pharmaceuticalproduct, feed, beverage, nutritional supplement, dairy product, food, orhealth food.

The composition may take different forms depending on the needs of thereceiver. In a preferred embodiment, the composition is in the form ofpowder, lozenge, granulation, microcapsule, ampoule/ampule, liquidspray, or suppository.

The composition of the invention can be used in animals or humans.Without affecting the effect of the active substance of Morchella, thecomposition comprising the active substance of Morchella can be madeinto any pharmaceutical form and administered to the animal or human ina suitable manner depending on the type of the drug.

Preparation of the Composition

Composition 1: The hot water extract (20 wt %) was taken as the activesubstance of Morchella, mixed well with benzyl alcohol (8 wt %) as apreservative, glycerin (7 wt %) as a diluent, and dissolved in purifiedwater (65 wt %) to produce a pharmaceutical composition of the presentinvention in liquid form. The aforementioned wt % means the ratio ofeach ingredient to the total weight of the composition. Store at 4° C.for later use.

Composition 2: The ethanol extract (15 wt %) was used as the activesubstance of Morchella, mixed well with benzyl alcohol (5 wt %) aspreservative, glycerin (10 wt %) as a diluent, and dissolved in purifiedwater (70 wt %) to produce a pharmaceutical composition of the presentinvention in liquid form. The aforementioned wt % means the ratio ofeach ingredient to the total weight of the composition. Store at 4° C.for later use.

What is claimed is:
 1. An active substance of Morchella for improvingdisturbance of reproductive function, prepared by the following methods:(A) inoculating Morchella mycelia on a medium for solid-state culture;(B) inoculating the Morchella mycelia cultured in step (a) in a liquidmedium for liquid culture; and (C) inoculating the Morchella myceliacultured in step (b) in a fermenter for fermentation.
 2. The activesubstance of claim 1, wherein a temperature of step (c) is 23 to 28° C.,the ventilation is 0.5 to 1.0 vvm, the rotation speed is 30 to 50 rpm,and/or the number of cultivation day is 6 to 10 days.
 3. The activesubstance of claim 1, wherein the Morchella is deposited in the FoodIndustry Research and Development Institute, with a deposit numberBCRC-36352 and/or BCRC-36336.
 4. A method for improving disturbance ofreproductive function, comprising an effective amount of an activesubstance of Morchella of claim
 1. 5. The method of claim 4, wherein theMorchella comprises Morchella esculenta, Morchella crassipes, or acombination thereof.
 6. The method of claim 4, wherein the activesubstance of Morchella is a lyophilized powder obtained afterfermentation and freeze-drying of mycelia of Morchella.
 7. The method ofclaim 4, wherein the active substance of Morchella is an extractobtained by extracting mycelia of Morchella with pure water or ethanol.8. The method of claim 4, wherein the reproductive function comprises atestosterone level, sperm type, oxidative stress in sperms, thestructural integrity of testicular tissues, or a combination thereof. 9.The method of claim 8, wherein the improving disturbance of reproductivefunction is increasing testosterone levels in the blood.
 10. The methodof claim 8, wherein the improving disturbance of reproductive functionis reducing the proportion of abnormal sperms with broken tails, benttails, or bent necks.
 11. The method of claim 8, wherein the improvingdisturbance of reproductive function is reducing the amount of ROSproduced in sperms to reduce the oxidative stress in sperms.
 12. Themethod of claim 8, wherein the improving disturbance of reproductivefunction is reducing the number and volume of gaps within the luminalcenter of the seminiferous tubules or between the seminiferous tubulesto improve the structural integrity of a testicular tissue.
 13. Themethod of claim 4, wherein the disturbance of reproductive function isinduced by obesity and/or metabolic syndrome.
 14. The method of claim13, wherein the metabolic syndrome is diabetes.
 15. A composition forimproving disturbance of reproductive function, comprising an effectiveamount of an active substance of Morchella of claim
 1. 16. Thecomposition of claim 15, wherein the active substance of Morchella is alyophilized powder obtained after fermentation and freeze-drying ofmycelia of Morchella.
 17. The composition of claim 15, wherein theactive substance of Morchella is an extract obtained by extractingmycelia of Morchella with pure water or ethanol.
 18. The composition ofclaim 15, further comprising an additive selected from the groupconsisting of an excipient, a preservative, a diluent, a filler, anabsorption enhancer, a sweetener, and a combination thereof.
 19. Thecomposition of claim 15, which is a medicine, feed, drink, nutritionalsupplement, dairy product, food, or health food.
 20. The composition ofclaim 15, which is in the form of powder, lozenge, granulation,microcapsule, ampoule, liquid spray, or suppository.